The Development and Opportunities of Predictive Biotechnology.

Recent advances in bioeconomy allow a holistic view of existing and new process chains and enable novel production routines continuously advanced by academia and industry. All this progress benefits from a growing number of prediction tools that have found their way into the field. For example, automated genome annotations, tools for building model structures of proteins, and structural protein prediction methods such as AlphaFold2TM or RoseTTAFold have gained popularity in recent years. Recently, it has become apparent that more and more AI-based tools are being developed and used for biocatalysis and biotechnology. This is an excellent opportunity for academia and industry to accelerate advancements in the field further. Biotechnology, as a rapidly growing interdisciplinary field, stands to benefit greatly from these developments.

A simple and robust LC-ESI single quadrupole MS-based method to analyze neonicotinoids in honey bee extracts.

Over the past years, neonicotinoids such as thiacloprid and flupyradifurone have gained considerable scientific and public interest. These molecules used as active compounds in pesticides are known due to cause drastic negative long-time effects on pollinators and even human health. Therefore, determining trace amounts of neonicotinoid in different environmental matrices by liquid chromatography coupled with mass selective detectors (LC-MS/MS or LC-Q-TOF/MS) has become an important methodology. However, not every scientific group has unlimited access to high-resolution mass-selective detectors (e.g., MS/MS). It becomes more apparent that the analytics of neonicotinoids are already a global issue. Research groups and organizations with a limited financial budget often depend on using cheap and robust equipment to do their analytical work. We demonstrate a single-quadrupole (Q) MS-based method with single-class residue methods (SRMs) for the analysis of neonicotinoids, applicable without the requirement of a high-end MS system. For an adequate sample clean-up strategy, QuEChERS (Quick, Easy, Cheap, Efficient, Rugged, safe) extraction and purification methods were modified and applied to eliminate residual matrix after honey bee extraction steps to analyze thiacloprid and flupyradifurone. •Simple liquid chromatography electro-spray ionization (LC-ESI) single-quadrupole mass selective (MS) method for neonicotinoid analysis.•Efficient sample pretreatment by a modified QuEChERS extraction and purification method.•Limit of detection (LOD) and limit of quantification (LOQ) for thiacloprid was 19.72 ng g-1 and 7.61 ng g-1, for flupyradifurone 65.73 ng g-1 and 25.36 ng g-1, respectively.

Exploiting Hydrogenophaga pseudoflava for aerobic syngas-based production of chemicals.

Gasification is a suitable technology to generate energy-rich synthesis gas (syngas) from biomass or waste streams, which can be utilized in bacterial fermentation processes for the production of chemicals and fuels. Established microbial processes currently rely on acetogenic bacteria which perform an energetically inefficient anaerobic CO oxidation and acetogenesis potentially hampering the biosynthesis of complex and ATP-intensive products. Since aerobic oxidation of CO is energetically more favorable, we exploit in this study the Gram-negative ß-proteobacterium Hydrogenophaga pseudoflava DSM1084 as novel host for the production of chemicals from syngas. We sequenced and annotated the genome of H. pseudoflava and established a genetic engineering toolbox, which allows markerless chromosomal modification via the pk19mobsacB system and heterologous gene expression on pBBRMCS2-based plasmids. The toolbox was extended by identifying strong endogenous promotors such as PgapA2 which proved to yield high expression under heterotrophic and autotrophic conditions. H. pseudoflava showed relatively fast heterotrophic growth in complex and minimal medium with sugars and organic acids which allows convenient handling in lab routines. In autotrophic bioreactor cultivations with syngas, H. pseudoflava exhibited a growth rate of 0.06 h-1 and biomass specific uptakes rates of 14.2 ±â€¯0.3 mmol H2 gCDW-1 h-1, 73.9 ±â€¯1.8 mmol CO gCDW-1 h-1, and 31.4 ±â€¯0.3 mmol O2 gCDW-1 h-1. As proof of concept, we engineered the carboxydotrophic bacterium for the aerobic production of the C15 sesquiterpene (E)-α-bisabolene from the C1 carbon source syngas by heterologous expression of the (E)-α-bisabolene synthase gene agBIS. The resulting strain H. pseudoflava (pOCEx1:agBIS) produced 59 ±â€¯8 µg (E)-α-bisabolene L-1 with a volumetric productivity Qp of 1.2 ±â€¯0.2 µg L-1 h-1 and a biomass-specific productivity qp of 13.1 ±â€¯0.6 µg gCDW-1 h-1. The intrinsic properties and the genetic repertoire of H. pseudoflava make this carboxydotrophic bacterium a promising candidate for future aerobic production processes to synthesize more complex or ATP-intensive chemicals from syngas.

Modulating proposed electron transfer pathways in P450BM3 led to improved activity and coupling efficiency.

Electrochemical in vitro reduction of P450 enzymes is a promising alternative to in vivo applications. Previously we presented three engineered P450BM3 variants for aniline hydroxylation, equipped with a carbon nanotube binding-peptide (CNT-tag) for self-assembly on CNT electrodes. Compared to wildtype P450BM3 the NADPH-dependent activity was enhanced, but the coupling efficiency remained low. For P450BM3 Verma, Schwaneberg and Roccatano (2014, Biopolymers 101, 197-209) calculated putative electron transfer pathways (eTPs) by MD simulations. We hypothesised that knockouts of these transfer pathways would alter the coupling efficiency of the system. The results revealed no improved system for the electrically-driven P450s. For the NADPH-driven P450s, however, the most active eTP-mutant showed a 13-fold increased activity and a 32-fold elevated coupling efficiency using NADPH as reducing equivalent. This suggests an alternative principle of electron transport for the reduction by NADPH and an electrode, respectively. The work presents moreover a tool to improve the coupling and activity of P450s with non-natural substrates.

The self-sufficient P450 RhF expressed in a whole cell system selectively catalyses the 5-hydroxylation of diclofenac.

P450 monooxygenases are able to catalyze the highly regio- and stereoselective oxidations of many organic molecules. However, the scale-up of such bio-oxidations remains challenging due to the often-low activity, level of expression and stability of P450 biocatalysts. Despite these challenges they are increasingly desirable as recombinant biocatalysts, particularly for the production of drug metabolites. Diclofenac is a widely used anti-inflammatory drug that is persistent in the environment along with the 4'- and 5-hydroxy metabolites. Here we have used the self-sufficient P450 RhF (CYP116B2) from Rhodococcus sp. in a whole cell system to reproducibly catalyze the highly regioselective oxidation of diclofenac to 5-hydroxydiclofenac. The product is a human metabolite and as such is an important standard for environmental and toxicological analysis. Furthermore, access to significant quantities of 5-hydroxydiclofenac has allowed us to demonstrate further oxidative degradation to the toxic quinoneimine product. Our studies demonstrate the potential for gram-scale production of human drug metabolites through recombinant whole cell biocatalysis.

Biooxidation of n-butane to 1-butanol by engineered P450 monooxygenase under increased pressure.

In addition to the traditional 1-butanol production by hydroformylation of gaseous propene and by fermentation of biomass, the cytochrome P450-catalyzed direct terminal oxidation of n-butane into the primary alcohol 1-butanol constitutes an alternative route to provide the high demand of this basic chemical. Moreover the use of n-butane offers an unexploited ubiquitous feed stock available in large quantities. Based on protein engineering of CYP153A from Polaromonas sp. JS666 and the improvement of the native redox system, a highly ω-regioselective (>96%) fusion protein variant (CYP153AP.sp.(G254A)-CPRBM3) for the conversion of n-butane into 1-butanol was developed. Maximum yield of 3.12g/L butanol, of which 2.99g/L comprise for 1-butanol, has been obtained after 20h reaction time. Due to the poor solubility of n-butane in an aqueous system, a high pressure reaction assembly was applied to increase the conversion. After optimization a maximum product content of 4.35g/L 1-butanol from a total amount of 4.53g/L butanol catalyzed by the self-sufficient fusion monooxygenase has been obtained at 15bar pressure. In comparison to the CYP153A wild type the 1-butanol concentration was enhanced fivefold using the engineered monooxygenase whole cell system by using the high-pressure reaction assembly.

New generation of biocatalysts for organic synthesis.

The use of enzymes as catalysts for the preparation of novel compounds has received steadily increasing attention over the past few years. High demands are placed on the identification of new biocatalysts for organic synthesis. The catalysis of more ambitious reactions reflects the high expectations of this field of research. Enzymes play an increasingly important role as biocatalysts in the synthesis of key intermediates for the pharmaceutical and chemical industry, and new enzymatic technologies and processes have been established. Enzymes are an important part of the spectrum of catalysts available for synthetic chemistry. The advantages and applications of the most recent and attractive biocatalysts--reductases, transaminases, ammonia lyases, epoxide hydrolases, and dehalogenases--will be discussed herein and exemplified by the syntheses of interesting compounds.

Recent progress in industrial biocatalysis.

In recent years, several procedures have been reported for the development of biocatalytic processes. This review focuses on selected examples integrating biocatalysts into a variety of industrially interesting processes ranging from the manufacture of smaller, chiral speciality chemicals to the synthesis of more complex pharmaceutical intermediates. The use of rational protein design, multistep processes and de novo design of enzyme catalysts for the stereocontrolled preparation of important target structures is discussed.